Paratuberculosis (Johne's disease) is caused by Mycobacterium avium subsp. paratuberculosis (Map), a facultative intracellular, acid-fast bacillus, and affects ruminants worldwide. In the United States, the disease causes the industry economic losses estimated at $200 and 250 million. The control of the disease is hampered by ineffective diagnostic methods, particularly in detection of sub-clinically infected animals.
A segment of infected animals in a given herd can be presumptively diagnosed based on clinical signs of diarrhea, emaciation, and/or serology. The animals can be reliably diagnosed with conventional and/or radiometric fecal culture. Detection of sub-clinically infected animals by serological and culture testing frequently leads to false negative results. Producers depend on “test-and-cull” programs to control the disease.
Several methods for screening for the presence of Map in tissue samples from affected animals are known. Commonly used immunological methods for detecting Map in a sample include agar gel immunodiffusion (AGID) tests and ELISA assays. More rapid DNA-based tests have been developed that utilize PCR in conjunction with pairs of primers that specifically detect species-specific insertion sequences present in Map strains, but not in other strains of Mycobacterium avium complex. A commercial DNA-based assay is available for detecting a 413 bp PCR product amplified from the Map insertion sequence defined as IS900 (Vary, P. H. et al., J Clin Microbiol 1990; 28:933-937, which is incorporated herein by reference in its entirety). When applied to the testing of milk for the detection of DNA of Map, the IS900-based PCR primers in commercial use do not identify the DNA of related pathogenic mycobacterium. To more completely prevent pathogenic mycobacteria from entering the human food supply through milk and milk-base products, the PCR primers used to evaluate milk for pathogenic mycobacteria would need to be more inclusive.
The current “gold standard” method for diagnosis of sub-clinical Map infection has been based upon fecal recovery of live Map using artificial culture media. Beckton-Dickinson Biosciences has recently developed an automated system (BACTEC MGIT 960 system) that can be used as a fully automated diagnostic tool for Johne's disease. Although this technique is highly specific, it is still suboptimal in terms of sensitivity. Additionally, culture from a fecal sample is only deemed negative after 49 days. This, however, is an imperfect diagnosis because cultures may become positive as long as six months after inoculation. In very rare instances, cultures have been reputed to become positive between six months and one year. Due to the amount of time a sample must be cultured, the expense of the specialized culture reagents and the BACTEC MGIT 960 system, this test is expensive. The cost to process a single sample ranges from $16.00 to $45.00 (depending upon the degree to which a given state subsidizes testing costs).
Map is not killed by pasteurization (1, 2). Viable Map and genomic variants enter the human food chain through milk and milk products. Mycobacterium avium subspecies paratuberculosis can be cultured from milk and selected cheeses (3-5) and constitutes the primary means by which Map and other pathogenic enteric mycobacterium enter into the human food supply.
Current testing using IS900 Map ELISA tests fail to identify up to one-third of cows shedding Map into their milk; Pinedo et al. found that 23.5% of cows with Map identified in their milk were deemed serologically negative for Map infection by IS900 ELISA Map tests. Another 11.8% had but a suspicious antibody titer (6). Wisziewska-Laszcych et al confirmed this initial report (7). The National Animal Health Monitoring System Study of 515 dairy farms demonstrated the presence of Map DNA in the bulk tank samples from 31.2% if the participating dairy farms.
In June 2001, the United Kingdom Food Standard Agency issued its report for food standards. The conclusion statement states “There is undoubtedly sufficient cause for concern (relative to Map as being the cause of Crohn's disease) for further action to be taken urgently to determine what the available data means . . . . This question can be divided into two areas: What action should be taken to reduce exposure to Map even though the causal link is not established; and what action can be taken to increase the knowledge base so that future decisions may be based upon more information (8).”
In 2008, the American Academy of Microbiologists published its report on Mycobacterium avium paratuberculosis: Infrequent human pathogen or public health threat (9). The executive summary states, “the association of MAP and CD is no longer in question. The critical issue today is not whether MAP is associated with CD, but whether MAP causes CD or is only incidentally present.”
By 2008, the majority of Koch's postulates for causation that can be ethically addressed had been effectively met (10-16). In 2009, three independent diagnostic laboratories (Michael T. Collins, Saleh A. Naser, and that of the Centers for Disease Control and Prevention) recovered Map from the blood of individuals with Crohn's disease (17). These three laboratories reaffirm the validity of Naser's previous recovery of Map from the blood of Crohn's patients as well as from the breast milk of two postpartum CD females without corresponding recovery from non-Crohn's diseased individuals.
From a medical infectious disease point of view, the validation of Naser's original findings cuts short the argument as to causality. If an individual has certain retroviruses in his or her white blood cells, he or she has HIV infection. If the individual has hepatitis B or C virus in his or her white blood cells, he or she has hepatitis infection. If an individual has Map in his or her white blood cells, he or she has infection with Map.
The natural history of Map infection/disease presumes a progressive three stage development. Classically, the pathogenesis of Johne's disease has been viewed as the progressive culmination of three stages of microbial involvement of the host animal Initial infection has been postulated to be acquired early and remain latent with or without intermittent evidence of fecal shedding until such time as serological evidence of infection can be detected (subclinical disease). Thereafter, the animals experience a progressive, chronic granulomatous infection that culminates in Johne's disease (17-18). What has been delineated in the literature is the progressive development of disease. The pathogenesis of Map induced disease is not the natural history of Map infection.
In developing the pathogenesis of Johne's disease in herbivores, three basic assumptions were made:                1. that Mycobacterium avium subspecies paratuberculosis (Map) is the cause, and not a cause, of Johne's disease;        2. that the IS900 insertion sequence is unique to Map isolates; and        3. that Mycobacterium avium complex (Mac) that includes Mycobacterium avium subsp. avium and M. hominissuis are environment and not pathogenic mycobacterium.         
Mycobacterium avium subspecies paratuberculosis (Map) is theorized to have evolved from Mycobacterium avium subsp. avium (Ma) (17-20). Map and Ma, by genetic criteria, are classified as subsets of the same species (20, 21). The literature on Johne's disease (chronic granulomatous enteritis in cattle) tends to deny the existence of pathogenic Map phenotypic variants more closely related to MA than to Map and that some mycobacterium are more Ma-like than Map-like (24-26). Genomic polymorphism is to be anticipated within species evolution. Such isolates are not identified by IS900 PCR primers. Darcel and Logen-Handsame have postulated that the failure of commercial Map ELISA tests to identify all clinically ill animals has been due to a lack of representation of the entire range of immunodominant test antigens (26).
IS1311 is present in Ma/Mac as well as Map. Primers based upon the IS1311 insertion sequence that identify Ma variants and Map are encompassed in the direct and nested fecal FecaMap® patented primers. The IS1311 insertion sequence is present in the vast majority of pathogenic mycobacterium. A long evolutionary time span is suggested by the presence of mutations in some of the IS1311 elements (17). None of the commercial Map ELISA tests including FUID#1 Map ELISA test have an antigen spectrum that identifies all potential pathogenic mycobacterium. 
A large Danish study demonstrated that declines in milk production attributable to Map occur over a long period of time and may not be realized without more advanced management tools (27).